Nazac André, Bancelin Stéphane, Teig Benjamin, Ibrahim Bicher Haj, Fernandez Hervé, Schanne-Klein Marie-Claire, De Martino Antonello
Department of Gynecology and Obstetrics, Bicêtre Hospital, Le Kremlin Bicêtre, France.
Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128, France.
Microsc Res Tech. 2015 Aug;78(8):723-30. doi: 10.1002/jemt.22530. Epub 2015 Jun 12.
Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the "gold standard" for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues.
偏振显微镜为各向异性样品提供了独特的信息。在其最完整的实现方式，即穆勒显微镜中，这项技术非常适合观察纤维状蛋白质的取向，首先是胶原蛋白。然而，未染色组织的固有光学各向异性必须通过天狼星红（PR）染色来增强，以便进行穆勒测量。在这项工作中，我们比较了穆勒显微镜和二次谐波产生（SHG）显微镜在PR染色的阴道和子宫颈组织样本上提供的取向映射。SHG是一种对纤维状胶原蛋白具有高度特异性的多光子技术，并被用作其可视化的“金标准”。我们表明，穆勒显微镜可以安全地用于确定PR染色宫颈组织中的胶原蛋白取向。相比之下，在阴道样本中，穆勒显微镜不仅揭示了胶原蛋白的取向，还揭示了其他各向异性结构的取向。因此，PR对胶原蛋白并不完全特异，这就需要在每种组织类型中与SHG显微镜进行比较。除了对PR特异性的这项研究外，我们还确定了染色参数的最佳值。我们发现，在宫颈和阴道组织中，5分钟的染色时间和5微米的样本厚度就足够了。
